Many molecular biology techniques depend on cloning individual cells from a mixture of cells.
For example, in the production of monoclonal antibodies, an essential step is hybridoma selection, including the separation and culture of individual hybridoma clones (fused myelomas and primary mouse cells). After cell fusion, the traditional way of selecting for monoclonality is to plate out single cells into 96-well dishes. This is repeated until clonality is assured.
Similarly, understanding gene function and identification of pharmaceutical leads requires the establishment of cell lines containing transfected genes expressed at an appropriate level. Standard techniques require the co-transfection of a gene with a dominant selectable marker followed by selection for growth for example in an antibiotic such as G418 or hygromycin. The resulting colonies are then picked by hand and further analysed for gene expression (RT-PCR) and functional expression.
Ascertaining optimal conditions for cell growth and differentiation requires broad testing of growth factors and culture conditions. The evaluation of a particular treatment requires a statistical approach on a large number of individual cells. One way to achieve this is to use numerous culture dishes, several for each treatment.
This process of cloning out may be modified and automated through the use of robots. Thus, for example, the ClonePix robot (manufactured by Genetix) implements this process by picking individual colonies directly from standard semi-solid media, the media preventing migration of the dividing cells. Thus, an imaging head captures images of colonies growing in the medium under white light, and software routines allow the separation and detection of individual colonies. A picking head then picks individual colonies into a 96-well plate.
Using a robot implemented picking method, colonies can be picked into 96-well plates at a picking speed of up to 400 clones per hour and graphic software allows the user to select colonies on the basis of size, shape, brightness and proximity. Furthermore, the software allows stratification of clones into slow, medium and fast growing cells, and clones of the same class may be grouped in the same 96-well plate. This gives rise to considerable savings in subsequent tissue culture steps as all wells can be processed at the same time.
However, the robot implemented cloning method relies on visualisation solely of colony size. Thus, the image capture only provides information on the size of the colony, and all colonies within a certain size range are picked. It is known for example that different hybridoma clones are capable of producing varying amounts of antibody. No information is provided or processed as to the productivity of different cells (i.e., the quantity of product produced or secreted), and the robot implemented cloning method therefore cannot discriminate between a high-producing hybridoma cell or colony and a low-producing hybridoma cell or colony. With regard to transfected cells, the robot cannot distinguish between clones with different levels of expression and/or secretion of recombinant protein.
A method disclosed in EP1752771 addresses this issue by identifying cells producing a polypeptide of interest using a combination of a class marker and a specificity marker. Marker-polypeptide complexes can then be detected, for example by an automated imaging system, and cells producing a high level of the polypeptide picked directly by a robot. However, this method necessitates the use of specific reagents such as antibodies to characterise production of the polypeptide of interest. This requires that such specific reagents are available and that different reagents must be used for detecting different polypeptides.
As mentioned above, selectable markers are often used in the identification of cell colonies expressing a protein of interest. It is also necessary to identify cell clones in which the vector sequences are retained during cell proliferation. In some cases stable vector maintenance is achieved either by use of a viral replicon or as a consequence of integration of the vector into the host cell's DNA.
It is often preferable to use amplifiable selectable markers when a high level of expression of a gene product is desired. The copy number of the vector DNA, and consequently the amount of product which is expressed, can be increased by selecting for cell lines in which the vector sequences have been amplified (e.g. after integration into the host cell's DNA).
A known method for carrying out such a selection procedure is to transfect a host cell with a vector comprising a DNA sequence which encodes an enzyme which is inhibited by a known drug. The vector may also comprise a DNA sequence which encodes a desired protein. Alternatively the host cell may be co-transfected with a second vector which comprises the DNA sequence which encodes the desired protein.
The transfected host cells are then cultured in increasing concentrations of the known drug thereby selecting drug-resistant cells. A common mechanism leading to the appearance of mutant cells which can survive in the increased concentrations of the otherwise toxic drug is the over-production of the enzyme which is inhibited by the drug. This most commonly results from amplification of vector DNA and hence gene copy number of the enzyme.
Where drug resistance is caused by an increase in copy number of the vector DNA encoding the enzyme, there is also an increase in the copy number of the vector DNA encoding the desired protein. There is thus an increased level of production of the desired protein.
The most commonly used system for such co-amplification uses dihydrofolate reductase (DHFR) as the enzyme. DHFR can be inhibited by the drug methotrexate (MTX). To achieve co-amplification, a host cell (which may lack an active gene which encodes DHFR) is transfected with a vector which comprises DNA sequences encoding DHFR and a desired protein. The genes for DHFR and desired protein may also be co-transfected into the cell on different vectors. The transfected host cells are cultured in media containing increasing levels of MTX, and those cell lines which survive are selected.
However, a disadvantage of such existing methods is that where an amplifiable selectable marker is used, the cells need to be grown for a time corresponding to a number of cell generations in order to adequately distinguish between transfected and non-transfected cells, or rather between cells having a high or a low selectable marker (e.g. DHFR) gene copy number. This is due to the time taken for untransfected cells to die and for cells showing amplification of the marker gene to outgrow those having a lower gene copy number. This produces a significant delay to the overall selection process. Typically, the selection process may take 6 months or more using such methods.
Therefore there is still a need for an improved method for detecting a cell producing a polypeptide of interest, which avoids the need for a lengthy growth phase during dominant marker selection and which provides a simple, rapid and widely-applicable selection procedure using readily-available reagents.